Double IHC question with clarification

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Hello again:
<p>Okay, so I didn't give enough information to get enough information.
<p>Linda, Patsy, Danielle: I know, <u>usually</u>
if they require the same pretreatment, there's no need for repeating
it. These guys need to have their own round of EDTA HIER :( . Believe
me, I've tried cutting one out, altering one's time, etc. I
have optimized them separately. Separately, they look great.
But I need to have the double staining on the same slide.
And that leads me to the next question.
<p>Cynthia: So why would anybody need to put two antibodies that
work best alone on the same slide? The answer is simply: pesky
Pathologist, pretty pictures for a publication, and a nice little challenge.
I should also mention that one antibody is a nuclear marker and the
other is a nice little membranous marker. Co-expression on the same
cell should make for some nice photos. And to answer your questions:
yes(substate), yes(times/temps), and no because that shows that you know
the many problem parameters of IHC.
<p>Diagnostically, most double immunostains are not practical.
For researchers it can serve many purposes (co-expression, working with
material limitations, antibody specificity, etc.). Also, most
Histotechs (especially those on the Histonet) like a challenge and
a beautifully stained slide. So, I'll continue to "tweak" my procedure
and wait for more responses or questions.
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<p>Thanks in advance,
<p>Jane Haley
<br>Barnes-Jewish Hospital
<br>St. Louis, Mo.
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